CXI Experimental Methods

Often, large crystals of a certain protein cannot be grown but a large number of very small crystals can readily be obtained. These sub-micron crystals do not scatter enough X-rays to yield an atomic structure using conventional protein crystallography techniques. The high flux of LCLS could allow these to be used for structure determination. This high flux/short pulse duration also has the advantage of allowing in principle damage-free higher resolution of radiation-sensitive samples such as metalloenzymes for example. Assuming all the nanocrystals possess the same crystal symmetry, a series of nanocrystals can be illuminated by LCLS X-ray pulses and the diffraction patterns recorded. A full 3D set similar to conventional protein crystallography can be assembled from many small crystals to determine the protein structure. The nanocrystals can be delivered into the CXI beam using a liquid jet (Weierstall et al. Rev. Sci. Instrum. (2010)), LCP jet (Weirstall et al. Nature Communications (2014)), an electrospinning jet (Sierra et al, Acta Cryst D (2012)) or mounted to a suitable substrate in vacuum (Frank et al. IUCrJ (2014)). The liquid jet options provides high data rate while the substrate mounting and LCP jet can help minimize sample consumption.

Small crystals also benefit kinetics and dynamics studies due to the higher activation fraction in small crystals. For both optically excited systems where penetration through the sample can limit the photoactivated yield and mix-and-inject systems where a substrate must diffuse through the crystal, small crystals are advantageous.

The CXI instrument is an in-vacuum high peak power instrument using hard X-rays. The vacuum environment provides extremely low background scattering which benefits gas phase samples. With proper alignment, the background can be less than a photon/data frame. Along with the ability to utilize higher photon energies and CXI’s short pulse UV laser capabilities, it provides an ideal environment to perform gas phase chemistry experiments – i.e. molecular movies that image chemical reactions in real time. 

The CXI instrument is primarily an in-vacuum high peak power instrument using hard X-rays. This makes CXI the ideal home for AMO science that requires the use of X-rays above 4-5 keV. Time-of-flight spectrometers and photon detectors can be added to the CXI sample chambers to allow AMO studies. 

The LCLS source produces hard X-ray fields of unprecedented high intensity. It allows for the first time tests of radiation damage models under such extreme conditions as well as tests of fundamental X-ray physics under extreme fields. These models are directly relevant to atomic-resolution imaging since the damage suffered during a pulse must be limited or the image reconstruction will suffer. The interaction of the powerful LCLS pulses with solid matter can be studied using the CXI instrument under unique extreme conditions using high-quality focusing optics.

It is possible, with the use of an optical pump laser, to obtain dynamic information of photo-induced changes in non-crystalline and crystalline samples with sub-picosecond time resolution. A flexible pump laser system (nanosecond and femtosecond lasers) is provided at the CXI instrument to allow a wide range of dynamics studies.

LCLS makes it possible to obtain two-dimensional projections of any non-reproducible nanoparticle and three-dimensional images of reproducible objects using the diffract-the-destroy technique. Furthermore, the CXI beam can be attenuated to a level slightly below the damage threshold of an inorganic nanoparticle and a full 3D reconstruction can in principle be obtained from a single particle using multi-image tomographic techniques. The transverse coherence length of the CXI beam can allow small particles to be imaged in 3D at high resolution.

The ultrashort pulses of LCLS open new possibilities to study short lived states of matter in extreme conditions. These extreme or transient states can be created with a pump laser and then probed with high time resolution with LCLS pulses using diffraction (Milathianaki et al. Science (2013)), scattering or in principle emission techniques.

Only a two-dimensional diffraction pattern can be collected from a single biomolecule before it is destroyed by the LCLS beam. Such a two-dimensional pattern encodes information about a projection image of the object onto a plane parallel to the detector. Three-dimensional structural information about highly-reproducible molecules such as viruses, large proteins or molecular complexes can be derived if a series of the molecules is delivered into the LCLS beam one after the other. Each molecule would have a different orientation and a full 3D diffraction data set can be obtained from a large number of identical copies of the sample. In theory, it could be possible to obtain high resolution structures for difficult to crystallize biomolecules. Research and development is required to demonstrate this capability.