Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

Abstract

The advent of the X-ray free electron laser (XFEL) in the last decade created the discipline of serial crystallography but also the challenge of how crystal samples are delivered to X-ray. Early sample delivery methods demonstrated the proof-of-concept for serial crystallography and XFEL but were beset with challenges of high sample consumption, jet clogging and low data collection efficiency. The potential of XFEL and serial crystallography as the next frontier of structural solution by X-ray for small and weakly diffracting crystals and provision of ultra-fast time-resolved structural data spawned a huge amount of scientific interest and innovation. To utilize the full potential of XFEL and broaden its applicability to a larger variety of biological samples, researchers are challenged to develop better sample delivery methods. Thus, sample delivery is one of the key areas of research and development in the serial crystallography scientific community. Sample delivery currently falls into three main systems: jet-based methods, fixed-target chips, and drop-on-demand. Huge strides have since been made in reducing sample consumption and improving data collection efficiency, thus enabling the use of XFEL for many biological systems to provide high-resolution, radiation damage-free structural data as well as time-resolved dynamics studies. This review summarizes the current main strategies in sample delivery and their respective pros and cons, as well as some future direction.

Recent Advances and Future Perspectives on Microfluidic Mix-and-Jet Sample Delivery Devices

“…An example of the lengthy 6-step fabrication procedure, shown in Figure 2, was described in detail by Calvey et al [27,28], which involves multiple flattening, polishing, tapering, and centring steps that require access to custom-designed chucks and jigs. However, using glass capillaries has the significant advantage of high pressure and solution pH resistance and, as a result, has been the most commonly used method for sample delivery for molecular imaging with XFEL [8,29]. [28]).…”

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

“…To overcome the sample consumption challenges, several sample conservation strategies for delivering crystals have been reported in the past years. Fortunately, smaller crystal size requirements and the ability to deliver crystals at room temperature have opened the door to new sample delivery methods for XFEL-based crystallography ( 52 ). Delivery of crystals on solid supports, also known as “fixed targets,” is one of the approaches whereby sample crystals are loaded on a matrix or a support device made from highly X-ray transparent materials such as silicon chips ( 53 , 54 ), thin membranes ( 55 , 56 ), elastomers ( 57 ), and thermoplastic materials ( 58 , 59 ), enabling in some cases more than 100% hit rates (multiple crystals per shot) as opposed to the typically low hit rates (1%–10%) currently achievable with continuous liquid delivery systems ( 6 , 60 , 61 , 62 ).…”

Segmented flow generator for serial crystallography at the European X-ray free electron laser

“…Droplet injection methods have the potential to overcome the limitations due to sample waste 32 , but in order to be compatible with MHz repetition rates of the EuXFEL they must achieve the fast replenishing requirements. In addition, any method for reducing sample waste, should also be compatible with time-resolved (TR) crystallography with the ultimate goal of constructing molecular movies 33 .…”

“…It is based on the generation of sub-nL crystal suspension droplets embedded in an immiscible oil, allowing injection with a traditional GDVN. This liquid injection method is based on creating fast jets in continuous mode injection suitable for MHz crystallography and will be compatible with currently available mix-and-inject approaches 23 , 32 . We demonstrate droplet generation of 3-deoxy-D- manno -octulosonate 8-phosphate synthase (KDO8PS) crystal suspensions with a microfluidic droplet generator and show that the droplet generation frequency can be controlled by the flow rates of the aqueous and oil streams.…”