Polyethylene Terephthalate Film-Based Sample Holder for Fixed-Target Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) enables the determination of macromolecular and chemical structures along with minimizing radiation damage. Sample delivery using the fixed-target (FT) scanning method minimizes both sample consumption and physical damage during delivery. When XFELs penetrate the FT sample holder, background scattering generated from the holder material can interfere with data processing, which consequently indicates the importance of selecting an appropriate FT sample holder material according to specific experimental conditions. We introduce a polyethylene terephthalate (PET) film-based sample holder that can be applied to data collection for both small-molecule and macromolecule XFEL diffraction studies. Background scattering analysis revealed that the PET film-based sample holder produced relatively low X-ray scattering in the high-resolution range (<10 Å) compared with a nylon mesh-based sample holder composed of polyimide and nylon mesh. Conversely, background scattering generated from the PET film was relatively higher in the low-resolution range (>10 Å) than that with the polyimide film; however, this low-resolution background was negligible for data processing. Thin PET films were manually perforated to promote the deposition of microcrystals in random orientations. The crystal samples were then deposited onto the films and sealed with an additional PET film to prevent dehydration. Using the PET film-based sample holder, we successfully collected diffraction data from barium titanate without unwanted background scattering from the sample holder. Furthermore, the room-temperature structure of glucose isomerase was determined using the PET film-based sample holder. Therefore, PET film-based sample holders can be applied to both small-molecule and macromolecular SFX data collection.