## 3 Single-Particle Imaging with X-ray Lasers
### Sample Delivery  
Sample delivery is a key component of XFEL experiments. There are two mainly used methods: (a) fix the sample by solid support, fixed target method; (b) Inject the particle streams into X-ray laser beam path, injection method. For the first method, Si\({}_{3}\)N\({}_{4}\) membrane or amorphous carbon film are regularly used. The method has been used for many 2D crystalline samples and synchrotron-based CDI experiments. However, the diffraction background is strong and can overwhelm the signal from the sample if Si\({}_{3}\)N\({}_{4}\) is used. For particle injectors, there are many injector devices commonly used by scientists. The gas dynamic virtual nozzle (GDVN) injector [173], delivers samples inside an unbroken liquid stream with tunable diameter by changing nozzle diameter and gas flow rate. The main general drawback of the GDVN is that they don't form droplets that are small enough. Also, non-volatile components in the buffer solution trend to stick to the particle of interest, which will make valid single particle shot more challenging. Another drawback of the GDVN is the high rate of sample consumption. This is not a problem when the sample is easy to achieve or synthesis. But this will be a big problem when the sample is valuable and hard to get. For SPI specifically, the liquid carrier medium scatters X-rays much more than the sample itself and makes it unsuitable for measuring single particle diffraction patterns of biological sampels. To solve this problem, the GDVN is always used combine with aerodynamic lens stack or electrospray during the SPI experiments.  
Another mainly used injector device is the aerosol injector. The particles can be focused by gradient pressure in the aerodynamic lens stack. The sample delivery method of choice for SPI is typically to transport the purified samples into the buffer solution and then aerosolized with helium gas using a GDVN and then introduced to the X-ray laser beam path via an aerodynamic lens. The purified samples could also be aerosolized by a gas nebulizer, such as electrospray [174], and then injected  
Figure 7: Projections of bioparticles used for SPI experiments. (**a**) Biological assembly of double-shelled rice dwarf virus (RDV). RDV is a member of the genus Phytoreovirus in the family Reoviridae. The atomic structure was determined by X-ray crystallography at 3.5 Å resolution [166]; (**b**) Quasi-atomic structure resolution model of bacteriophage PRD1 wild type virion. PR772 belongs to the PRD1 family. The dsDNA bacteriophage has a membrane inside its icosahedral capsid. The atomic structure was determined by combined cryo-EM and X-ray crystallography at 25 Å resolution [169]; (**c**) Crystal structures of MS2 coat protein mutants in complex with wild-type RNA operator fragments. MS2 is a group I RNA bacteriophage that infects _Escherichia coli._ The atomic structure was resolved by X-ray crystallography at a resolution of 2.8 Å [170]. The projections were generated by NSL Viewer, a WebGL based 3D viewer [171; 172].  
by the aerodynamic lens stack [175]. These unfortunately typically lead to low hit rates. For CXI nanofocusing, the hit rate is just \(\sim\)1% or lower. The number of diffraction patterns needed for high resolution is large, requiring a lot of beamtime, beyond what is reasonably possible. This is an issue that will need to be resolved for SPI to become a broadly useful method. Another challenge for SPI is the scattering background from residual gas of aerosol injector. Different gases, such as He and CO\({}_{2}\), have been tested and there are indications that the background level is now limited by the aerosol carrier gas.