Micro-patterned polymer-based fixed targets for serial crystallography at synchrotrons and XFELs
Serial crystallography at X-ray free electron lasers (XFELs) and synchrotron light sources, named serial femto-second crystallography (SFX) and serial synchrotron crystallography (SSX), respectively, has proved to be a successful and robust methodology for determining the structures of macromolecules at room temperature or near physiological temperatures with minimal radiation damage.To cater for these different experiments, a wide variety of delivery methods have been developed [1,2].Amongst these, fixed-targets, based on micro-pattern solid-supports or chips [3] and precise stage-motion [4], have proved to be a strong and dependable approach.Fixed-target sample delivery methods allow for a reduction of sample consumption and rapid optimization of sample loading parameters.Fixed-targets also lend themselves to high throughput technologies and an increased ability to locate and position crystals.Silicon wafers are the most common fixed-target and offer an inert support for the immobilized crystals and a precise aperture array for rapid alignment strategies [4].However, the silicon wafers are highly brittle, prone to fracture and are highly expensive.They are also opaque, making it difficult to know a priori how well crystals have been loaded into the apertures and they can give strong Si(111) reflections if misaligned [5,1].Given these issues, it is worthwhile pursuing alternative materials as the basis for these micro-pattern supports.Here we present our micro-pattern, thin-film polymer based fixedtarget developed for TR-SFX using the SwissMX endstation at the Cristallina experimental station of SwissFEL [Fig.1].Using silicon microfabrication and polymer replication technologies, we have designed inverted pyramidal shaped wells in membranes ranging from 25-50 µm in thickness.This design enables crystals to funnel into predefined positions, optimizing the hit-rate of the probing X-ray beam.The polymer-based film provides low x-ray absorption and scattering background, high design flexibility and the potential for mass-fabrication at low cost.Here we present the methodology for the manufacture of this fixed-targets and a summary of their use at Cristallina for both standard SFX and time-resolved experiments as well as preliminary data from the timeresolved binding of photocaged biotin to streptavidin.Figure 1. A. SwissMX fixed-target endstation at the Cristallina experimental station of SwissFEL.B. Micro-patterned, polymer-based SwissMX fixed-target composed of inverted pyramidal membrane, fiducials, and a labelling system.
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