Sample Supports

Sample-support films - "Thin films to support crystals were microfabricated in polyimide using MiTeGen’s standard processes. These allow the prototyping and production of multiple designs with low up-front design and tooling cost and low incremental production cost. Polyimide is optically and X-ray transparent, mechanically tough at room and cryogenic temperature, and radiation-hard for a polymer, and is widely used for X-ray sample holders and windows. Mechanical toughness and X-ray damage resistance are essential for sample holders to be reusable. Polyimide has strong optical absorption below 380 nm, giving it a gold color. This absorption does not hinder visible-light microcrystal imaging as long as the films are thin (<30 mm); it affects UV excitation fluorescence imaging in transmission but not in epi mode, and it does not affect two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) imaging at typical excitation wavelengths near 1064 nm.

Crystal sizes and shapes are diverse, and diffraction-quality crystals may be abundant or scarce. Locating crystals at well defined positions on the sample support film allows step-and repeat data collection, reducing data-acquisition times compared with continuous raster scanning.

Sample-support frame -"The sample-support films (Fig 2) were bonded using a thermally activated epoxy to a 250 or 500 mm thick frame. The film was attached so that its crystal-holding active area faced into an aperture in the frame. The frame protects crystals from contact with sealing films (Section 2.1.4) and provides additional depth (beyond that of wells patterned into the support film) to retain liquid, which is useful in crystal repositioning and for in situ crystallization."

Goniometer base - A magnetic steel goniometer base is modified to capture the frame. There are 3 types of bases available which are X, Y and Z. Custom bases are also available upon request. Bases are designed to allow the easy separation of frames and bases for storage, in situ crystallization, cleaning, reuse and recycling.

For room-temperature data collection, the sample support must be sealed to minimize dehydration prior to and during X-ray data collection. The experiments in Section 4.3 led to the use of a 3.2 mm thick Mylar sealing film which was attached to the frame using a thin laser-cut double-sided adhesive gasket. For room-temperature storage for hours to days (sufficient for transport to a synchrotron as in the present experiments at NSLS-II), the sealed sample film plus frame plus goniometer base was placed in a modified magnetic cryovial containing an absorbent polymer plug soaked with, for example, reservoir solution or crystallization buffer. For longer term storage, commercial in situ crystallization and storage trays accepting goniometer-base-mounted sample supports, based on the designs of Baxter et al. (2016), can be used. Alternatively, the sealed sample film plus frame can be removed from the goniometer base and stored in, for example, an Eppendorf tube containing a solution-soaked absorbent polymer plug or in a custom-designed in situ crystallization and storage tray.

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