# Upgraded Combined Inject-and-Transfer System for Serial Femtosecond Crystallography
## 1 Introduction  
The traditional macromolecular crystallography (MX) technique enables the determination of crystal structure at atomic resolution [1, 2]. MX helps in elucidating the molecular mechanism of macromolecules, provides insight into drug design, and enables rational engineering of industrially applicable enzymes [3, 4, 5]. However, traditional MX has experimental limitations, such as the occurrence of radiation damage and the necessity for cryogenic environments, and it is limited to static structural information [6]. These technical limitations can be overcome with serial crystallography (SX) using X-ray free-electron lasers (XFEL) or synchrotron X-rays [7]. The SX technique enables the determination of room-temperature structures with minimal radiation damage, which provides more accurate data on the structural fluctuations of target molecules [8, 9, 10]. Moreover, time-resolved molecular dynamics can be observed via pump-probe or mix-and-injection experiments [11, 12, 13]. In an SFX experiment, the crystal sample is exposed to XFEL only once. To collect the complete dataset, numerous microcrystals are continuously delivered to the X-rayinteraction point. To deliver a large number of microcrystals, various sample delivery methods, such as injectors [14; 15; 16], viscous media [17; 18; 19; 20; 21; 22; 23; 24], fixed-target scanning [25; 26; 27; 28; 29; 30; 31], capillary methods [32; 33], microfluidic devices [34; 35; 36; 37], and hybrids, have been developed and applied in SX data collection. The NCI experiment hutch at the Pohang Accelerator Laboratory X-ray Free-Electron Laser (PAL-XFEL) facility has two SFX sample chambers to deliver microcrystals to the X-ray interaction point: (1) a multifarious injection chamber for molecular structure study (MICOSS) [38] and (2) fixed-target SFX (FT-SFX) chambers. In the MICOSS system, particle solution delivery (PSD) [38], carrier matrix delivery (CMD) [38], and microliter volume (MLV) syringe injectors [39] have been applied to deliver microcrystals into the XFEL beam path in helium or air environments. This system allows for the collection of general XFEL diffraction data and pump-probe experiments using optical lasers. In the FT-SFX system, a fixed-target approach using a conventional 2D raster (with a nylon mesh sample holder or viscous medium-based crystal support) [28; 30] or 1D scanning using polyimide tubing with a servo view system [31] can be performed. Among these sample delivery methods, the fixed-target scanning system has the advantage of minimizing the consumption of crystal samples, as well as the physical damage to crystals, during sample delivery. When a fixed-target sample holder is exposed to an XFEL, bubbles are generated from the crystal suspension, indicating that radiation damage has occurred and radicals may be generated. Although it has not been experimentally proven how radiation damage due to initial XFEL exposure affects the samples, it is prudent to minimize radiation damage. From this perspective, the injector system has advantages as it continuously releases fresh samples from the injector. However, this system requires more effort to create a stable injection stream, and sample consumption is higher than with the FT-SFX system. We recently developed a combined inject-and-transfer system (BITS) and successfully applied it to data collection [40]. Crystal suspensions or crystals embedded in a viscous medium are deposited on UV/ozone (UVO)-treated polyimide film using an injector needle and transferred to the X-ray interaction point in a programmed translation stage. As all crystal samples excluded from the injector are deposited on the UVO-treated polyimide film, establishing a stable injection stream is not essential, which allows for improved beamtime efficiency [40]. Typically, while the width of the general injection stream from the injector or syringe is much larger than the XFEL beam size, X-rays are only transmitted to one point of the injection stream, thereby wasting unexposed samples.