3D printed droplet generation devices for serial femtosecond crystallography enabled by surface coating

Abstract

The role of surface wetting properties and their impact on the performance of 3D printed microfluidic droplet generation devices for serial femtosecond crystallography (SFX) are reported. SFX is a novel crystallography method enabling structure determination of proteins at room temperature with atomic resolution using X‐ray free‐electron lasers (XFELs). In SFX, protein crystals in their mother liquor are delivered and intersected with a pulsed X‐ray beam using a liquid jet injector. Owing to the pulsed nature of the X‐ray beam, liquid jets tend to waste the vast majority of injected crystals, which this work aims to overcome with the delivery of aqueous protein crystal suspension droplets segmented by an oil phase. For this purpose, 3D printed droplet generators that can be easily customized for a variety of XFEL measurements have been developed. The surface properties, in particular the wetting properties of the resist materials compatible with the employed two‐photon printing technology, have so far not been characterized extensively, but are crucial for stable droplet generation. This work investigates experimentally the effectiveness and the long‐term stability of three different surface treatments on photoresist films and glass as models for our 3D printed droplet generator and the fused silica capillaries employed in the other fluidic components of an SFX experiment. Finally, the droplet generation performance of an assembly consisting of the 3D printed device and fused silica capillaries is examined. Stable and reproducible droplet generation was achieved with a fluorinated surface coating which also allowed for robust downstream droplet delivery. Experimental XFEL diffraction data of crystals formed from the large membrane protein complex photosystem I demonstrate the full compatibility of the new injection method with very fragile membrane protein crystals and show that successful droplet generation of crystal‐laden aqueous droplets intersected by an oil phase correlates with increased crystal hit rates.

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

“…1, where the droplets generated in a microfluidic droplet generator are shown schematically in a capillary from which they are jetted to interact with the EuXFEL X-ray pulses. To integrate this principle in a typical SFX liquid injection setup employing a GDVN to deliver the sample in a vacuum chamber, we employed a 3D printed microfluidic droplet generator, as previously described 44 . Here, we adapted this approach for a workflow compatible with the early user experiments at the EuXFEL as depicted in Fig.…”

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

“…Another approach utilized the immiscibility between water and oil. In this "water-in-oil" approach, the crystal stream is mixed into oil at regular interval using a 3D-printed droplet generator [163]. The resulting oil stream, with crystals encapsulated in distinct oil droplets, is then ejected into the XFEL pulse using a GDVN nozzle [163].…”

“…In this "water-in-oil" approach, the crystal stream is mixed into oil at regular interval using a 3D-printed droplet generator [163]. The resulting oil stream, with crystals encapsulated in distinct oil droplets, is then ejected into the XFEL pulse using a GDVN nozzle [163]. Smaller nanoliter-sized droplets have also been generated from microfluidic devices using surface acoustic waves (SAW) [164].…”

High-throughput in situ experimental phasing

“…However, very high quality diffraction data from significantly smaller crystals, including submicrometresized samples, are often achievable using SFX methods (Gati et al, 2017;Nass, Redecke et al, 2020). Consequently, various sample-presentation strategies have been developed to minimize background noise for SFX, because nanometre-sized to micrometre-sized crystals produce weaker diffraction data compared with traditionally sized samples (Awel et al, 2018;Beyerlein et al, 2017;Calvey et al, 2019;Dasgupta et al, 2019;Davy et al, 2019;Doak et al, 2018;Echelmeier et al, 2019;Fuller et al, 2017;Grunbein & Nass Kovacs, 2019;Lieske et al, 2019;Martiel et al, 2019;Meents et al, 2017;Mehrabi et al, 2019;Monteiro et al, 2019;Nass, Gorel et al, 2020;Nogly et al, 2016;Oberthuer et al, 2017;Orville, 2017;Owen et al, 2017;Roedig et al, 2017;Schulz et al, 2018Schulz et al, , 2019Shelby et al, 2020;Sierra et al, 2016;Stagno et al, 2017;Suga et al, 2020;Sugahara et al, 2017;Weinert et al, 2017;Wiedorn et al, 2018;Zhao et al, 2019). In addition, X-ray pulse durations of tens of femtoseconds and the tight focus and intensity of XFEL pulses often produce very high-quality data sets with little or no radiation-induced effects in the data and refined atomic models.…”