# Upgraded Combined Inject-and-Transfer System for Serial Femtosecond Crystallography
## 3 Results and Discussion
### Sample Injection Experiment  
In a previous study, we investigated the deposition of crystallization solution (100 mM Na-acetate, pH 4.5, 5% (\(w/v\)) PEG 8000, and 3.5 M NaCl) on polyimide films during inject-and-transfer development [40]. The crystallization solution was not deposited stably because of the hydrophobic properties of the polyimide film, which formed a drop of solution at the needle tip. To solve this problem, the hydrophilicity of the polyimide film surface was increased by UVO treatment. Then, the crystallization solution was stably deposited [40]. The surface of the polyimide film subjected to UVO treatment has a disadvantage in that the hydrophilicity decreases when exposed to air. Thus, avoiding long-term exposure to air after UVO treatment is critical. Accordingly, it is necessary to continuously replace the UVO-treated film after a certain period, which is inconvenient and lowers beamtime efficiency. To solve this problem, we considered the direct use of non-UVO-treated hydrophobic polyimide films by increasing the sample viscosity, which has the advantage of simplifying the experimental process for UVO treatment. Figure 1: BITS system for SFX experimentation. (**A**) Schematic of the BITS system containing the translation stages for sample injections and target scanning. (**B**) Photo of current BITS system in the FT-SFX chamber. (**C**) Close-up view of \(XYZ\)-axis motorized piezo stage for alignment between syringe needle and polyimide film. Figure 2: BITS control software GUI. GUI programs for (**A**) fixed-target control and (**B**) syringe needle position control. From the perspective of protein samples, typical crystal suspensions may exhibit low viscosity and hydrophilic properties. In contrast, crystallization solutions often contain highly viscous or hydrophobic substances. We expected that a crystallization solution containing a highly viscous substance would deposit well owing to the hydrophobic properties of polyimide film surfaces. Glycerol is used to reduce protein aggregation during purification and storage. In addition, glycerol is widely used as a cryoprotectant solution in traditional macromolecular crystallography experiments. Accordingly, we considered that there would be stable crystal samples even when glycerol was added to the crystal suspension. Polyethylene glycol (PEG)-based reagents are widely used as precipitants in protein crystallization and exhibit high viscosity at specific concentrations. Considering this, we investigated the deposition of glycerol and polyethylene glycol solutions on polyimide films. If a high concentration of glycerol or PEG is used, the viscous solution will deposit well on the polyimide film, but there may be physical damage to the crystals during long-term incubation. Accordingly, the concentrations of glycerol or PEG solution used in this experiment were \(\leq\)30%. All solutions were delivered onto the polyimide film through a syringe needle at flow rates of 0.5, 1, and 10 nL/min. When 10% (\(v/v\)) glycerol and 10% (\(v/v\)) PEG 400 were injected, it was observed that a drop was formed at the injection needle tip without being deposited on the polyimide film (Figure 3A,B). This indicates that the injected glycerol and PEG 400 have low viscosity when interacting with the hydrophobic surface of the polyimide film. The concentrations of glycerol and PEG 400 were increased to 20% or 30% to increase viscosity. The 20% and 30% (\(v/v\)) glycerol solutions were not stably deposited on the polyimide film (Figure 3A). These formed a drop at the tip of the needle tip and, as the size increases with continuous sample injection, irregularly shaped large drops were seated on the polyimide film. For PEG 400, the 20% (\(v/v\)) PEG 400 solution was not deposited on the film, whereas the 30% (\(v/v\)) PEG 400 solution was continuously deposited on the polyimide film at 1 and 10 nL/min (Figure 3B). However, the shape of the deposited solution was irregular and the experimental reproducibility was low (Figure 3B).