*3.3. Background Scattering*

Resolution is used as an important parameter for evaluating the quality of diffraction data and analyzing the effect of background scattering. The highest-resolution shells are determined using the following criteria: signal-to-noise ratio [I/σ(I)] > 2, redundancy > 2, completeness > 85%, and Rmerge < 1.

The background scattering of the crystal support material will affect the quality of the diffraction data of the protein crystal. When the background scattering of the support material is large, it will increase the noise points of the diffraction data, thereby masking the signal intensity of the diffraction points of the protein crystal. The selection of the material of the crystal support film requires special consideration: (1) High temperature resistance; high temperature will be generated after X-ray irradiation of the film material, which is likely to cause the film of some materials to be burnt and deformed. (2) High transparency; thin film materials with low light transparency are not conducive to the positioning of microcrystals. Thin films with high transparency can make the crystals visible on the microscope on the beamline station, which can efficiently locate the crystal position and facilitate data collection. (3) Chemical corrosion resistance; the crystal solution may contain corrosive components, or may easily chemically react with some thin film materials, resulting in the thin film materials being unusable. (4) Radiation resistance; synchrotron radiation X-ray has a high luminous flux, so the radiation dose is large, and the selected thin film material should be radiation resistant. At present, the commonly used films for serial crystallographic experiments based on fixed targets include Maylar film, polyimide film, synthetic cyclic olefin copolymer (COC), polycarbonate plastic, and other materials. Mylar film is a kind of polyester film with good light transmittance. The COC film is resistant to high temperature and chemical corrosion. Polyester film has a high melting point and good light transmission performance. The polyimide membrane, for example Kapton membrane, has many advantages and is best suited to all the requirements of our experiment, it is a kind of membrane with high temperature resistance, good light transmission performance, and chemical corrosion resistance. When the energy is 9–15 keV, the X-ray transmittance of 12.5 μm thick polyimide film can reach 99%, and it produces low background scattering under X-ray irradiation. Polyimide film has very good light transmittance, and microcrystals of approximately 10 μm are visible under the microscope. Therefore, it is very easy to locate the position of the crystal at the beamline station. Here, we only use Kapton membrane for our experiment.

In order to explore the influence of the background scattering of the in situ device including the crystallization drops and Kapton membranes on the quality of the protein crystal diffraction data, the air diffraction image (Figure 4a) and the in situ device diffraction image (Figure 4b) were collected respectively. Comparing the background scattering of air with the in situ device, it can be found that there is little difference between the two experiments (Figure 4a,b). In order to further prove the influence of the in situ device on the analysis of diffraction data, we collected the diffraction data of microcrystals by using the in situ device and a nylon loop. From the results of the resolution signal-to-noise ratio data (Figure 4c,d). It can be seen that there is little difference between the in situ device and the nylon loop. The in situ diffraction device has a major influence on the signal-to-noise ratio of the crystal diffraction data at approximately 4 Å, but the difference is basically negligible. The results show that the method of loading a microcrystal with the in situ device can also obtain high-quality diffraction data. The in situ device has lower background scattering and will not affect the quality of the crystal data.

**Figure 4.** Background noise of air (**a**) and the in situ device (**b**) derived from the same coordinate point of the two diffraction patterns. (**c**) Signal-to-noise ratio in every resolution shell of the diffraction data obtained from a single lysozyme crystal in the nylon loop. (**d**) Signal-to-noise ratio in every resolution shell of the diffraction data obtained from multiple lysozyme crystals in the microplate.
