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Serial crystallography (SX) provides room‐temperature crystal structures with minimal radiation damage and facilitates the comprehension of molecular dynamics through time‐resolved studies. In SX experiments, it is important to deliver a large number of crystal samples to the X‐ray interaction point in a serial and stable manner. The advantage of crystal delivery in a viscous medium via a capillary is the ability to deliver all of the crystal samples to the X‐ray interaction point at a low flow rate; however, the capillary often breaks during handling and high X‐ray absorption can occur at low energy states. This study aimed to develop a stable system for sample delivery in a viscous medium via a polyimide‐based single‐channel microfluidic (PSM) chip for SX. Since this microfluidic chip comprises a polyimide film, it has high tensile strength and higher X‐ray transmittance than a quartz capillary. The PSM chip was connected to a syringe containing the microcrystals embedded in viscous medium. The channel of the PSM chip was aligned to the X‐ray path, and the viscous medium containing lysozyme crystals was stably delivered using a syringe pump at a flow rate of 100 nl min−1. Room‐temperature lysozyme crystal structures were successfully determined at 1.85 Å resolution. This method would greatly facilitate sample delivery for SX experiments using synchrotron X‐rays. [ABSTRACT FROM AUTHOR]

Serial femtosecond crystallography (SFX) enables the determination of the room‐temperature crystal structure of macromolecules, as well as providing time‐resolved molecular dynamics data in pump–probe experiments. Fixed‐target SFX (FT‐SFX) can minimize sample consumption and physical effects on crystals during sample delivery. In FT‐SFX studies, having a sample holder that can stably fix crystal samples is one of the key elements required for efficient data collection. Hence, development of sample holders from new materials capable of supporting various crystal sizes and shapes may expand the applications of FT‐SFX. Here, a viscous‐media‐based crystal support in a sample holder for FT‐SFX is introduced. Crystal samples were embedded in viscous media, namely gelatin and agarose, which were enclosed in a polyimide film. In the vertically placed sample holder, 10–15%(w/v) viscous gelatin and 1–4%(w/v) agarose gel stably supported crystals between two polyimide films, thereby preventing the crystals from descending owing to gravity. Using this method, FT‐SFX experiments were performed with glucose isomerase and lysozyme embedded in gelatin and agarose, respectively. The room‐temperature crystal structures of glucose isomerase and lysozyme were successfully determined at 1.75 and 1.80 Å resolutions, respectively. The glucose isomerase and lysozyme diffraction analyses were not impeded by excessive background scattering from the viscous media. This method is useful for delivering crystal samples of various sizes and shapes in FT‐SFX experiments. [ABSTRACT FROM AUTHOR]