# Upgraded Combined Inject-and-Transfer System for Serial Femtosecond Crystallography
## 3 Results and Discussion
### Preliminary Study for Inject-and-Diffuse in BITS  
In nature, numerous biomolecules, such as proteins and nucleic acids, interact with substrates (or inhibitors) and cleave, synthesize, or modify them. Observation of structural reaction changes is useful for understanding the exact molecular mechanism. By mixing the protein crystal and the substrate (or inhibitor) solution and collecting diffraction data at various mixing times, the entire reaction mechanism between the protein and the substrate can be dissected. Various liquid application-based time-resolved SX experiments, such as mix-and-inject [48] and drop-on-drop [49], have been developed. These methods are effective when a rapid reaction occurs between the protein and the substrate (or inhibitor) solution. However, time-resolved SFX experiments using liquid applications for slow reactions have not been actively studied. We performed a preliminary inject-and-diffuse experiment using a BITS system during XFEL beamtime. The basic experimental concept of the inject-and-diffuse approach is similar to that of the BITS system using the inject-and-transfer method. In the inject-and-diffuse approach in BITS, two injectors deposit two different samples on a polyimide film  
Figure 3: Injection experiment with viscous solutions on polyimide film in BITS: (**A**) 10–30% (\(v/v\)) glycerol solution; (**B**) 10–30% (\(v/v\)) PEG 400 solution; (**C**) 10% (\(w/v\)) PEG 3350; and (**D**) 10% (\(w/v\)) PEG 6000 solutions. in the same manner as in BITS. The crystal suspension is injected from one syringe needle, and the inhibitor solution is injected from the other syringe needle (Figure 4A). The crystal suspension is first deposited on the film and translates the film to the XFEL position; then, the crystal suspensions meet the inhibitor solution, which is mixed and diffused, and it is moved to the XFEL position by the translation stage (Figure 4A). The time delay for time-resolved experiments can be adjusted as desired by adjusting the speed of the translator or of the interval between the mixing and XFEL positions. To demonstrate the inject-and-diffuse experiment in BITS, lysozyme crystals were embedded in a lard viscus medium to prevent crystal clogging in tubing or syringe needles. This crystal sample was deposited on a UVO-treated polyimide film using a syringe needle, and a lysozyme inhibitor solution was added to the crystal sample via injection from the second syringe needle (Figure 4B). We obtained diffraction images from this preliminary experiment for the inject-and-diffuse approach (Figure 4C), but complete datasets were not obtained because of the limited XFEL beamtime and crystal clogging issue. As a result  
Figure 4: Preliminary data for inject-and-diffuse approach in the BITS system. (**A**) Schematic drawing of the sample loading in the inject-and-diffuse approach. (**B**) Schematic of the inject-and-diffuse system in the BITS. This experiment was performed before upgrading the injection needle stage; thus, the manual XYZ stage was installed. (**C**) Snapshot of the inject-and-diffuse experiment during XFEL data collection. (**D**) Indexing of the XFEL diffraction image from a preliminary inject-and-diffuse experiment. The predicted Braggs peak positions are indicated by circles, which were generated by the CrytFEL program. of indexing the diffraction images obtained from inject-and-diffuse experiments in BITS, crystals belonging to the tetragonal P4\({}_{3}\)2\({}_{1}\)2 space group and unit cell dimensions for a, b and c were 77.69 A, 77.69 A and 38.14 A, respectively. These unit cell parameters for a and b were smaller when compared to a previously reported lysozyme crystal structures from BITS applications (P4\({}_{3}\)2\({}_{1}\)2, a = b = 79.48 A and c = 38.48 A). We observed the electron density map of lysozyme using the incomplete dataset. Although we could not determine the time-resolved structural change, this preliminary experiment demonstrates the potential of applying the mix-and-diffuse approach in the BITS system.