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. 2024 Feb 21;11(1):011101.
doi: 10.1063/4.0000229. eCollection 2024 Jan.

Advanced manufacturing provides tailor-made solutions for crystallography with x-ray free-electron lasers

Lars Paulson  1 , Sankar Raju Narayanasamy  2 , Megan L Shelby  2 , Matthias Frank, Martin Trebbin
Affiliations

Advanced manufacturing provides tailor-made solutions for crystallography with x-ray free-electron lasers

Lars Paulson et al. Struct Dyn. .

Abstract

Serial crystallography at large facilities, such as x-ray free-electron lasers and synchrotrons, evolved as a powerful method for the high-resolution structural investigation of proteins that are critical for human health, thus advancing drug discovery and novel therapies. However, a critical barrier to successful serial crystallography experiments lies in the efficient handling of the protein microcrystals and solutions at microscales. Microfluidics are the obvious approach for any high-throughput, nano-to-microliter sample handling, that also requires design flexibility and rapid prototyping to deal with the variable shapes, sizes, and density of crystals. Here, we discuss recent advances in polymer 3D printing for microfluidics-based serial crystallography research and present a demonstration of emerging, large-scale, nano-3D printing approaches leading into the future of 3D sample environment and delivery device fabrication from liquid jet gas-dynamic virtual nozzles devices to fixed-target sample environment technology.

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Conflict of interest statement

The authors have no conflicts to disclose.

Figures

FIG. 1.
FIG. 1.
Overview of various 3D printing efforts for serial crystallography at XFELs and synchrotrons. (a) Two-photon polymerization (2PP)-based gas-dynamic virtual nozzles (GDVN) with mixing elements [Reproduced with permission from J. Knoška, et al., Nat. Commun. 11, 657 (2020). Copyright 2020 Authors, licensed under a Creative Commons Attribution (CC BY) license]; (b) Projection microlithography-based hydrodynamic flow focusing chip for serial crystallography experiments [Reproduced with permission from D. C. F. Monteiro et al., IUCrJ 7, 207 (2020); Copyright 2020 Authors, licensed under a Creative Commons Attribution (CC BY) license]; (c) 2PP fabrication-based extrusion nozzle for Lipidic Cubic Phase (LCP) samples [Reproduced with permission from M. Vakili et al., J. Appl. Crystallogr. 56, 1038 (2023). Copyright 2023 Authors, licensed under a Creative Commons Attribution (CC BY) license]; (d) 2PP fabrication-based flat jet nozzle [Reproduced with permission from P. E. Konold et al., IUCrJ 10, 662 (2023). Copyright 2023 Authors, licensed under a Creative Commons Attribution (CC BY) license]. 2PP fabrication has significantly advanced the sample environment and delivery for serial crystallography experiments.
FIG. 2.
FIG. 2.
Rapid large-area two-photon polymerization: (a) Schematic representation of large-area 2PP: here the features are enlarged for artistic rendition. The femtosecond laser-based rapid large-area 2PP instrumentation schematic representation. This setup has a very large area of up to 160 × 160 mm2 with the ability to print intricate sub-micron features. (b) Printing demonstration of the new rapid large region 2PP setup with the features required for fixed-target sample delivery, i.e., a minimum length of 500 μm and features smaller than 5 μm. As a demonstration, sub-micron featured structure is printed over a very long range of 2.5 cm (compared to a penny). The layer thickness can be custom adjusted on-demand for each feature, down to the sub-micron range. In these examples, the layer thickness is greater for visibility. (c) The printing ability is shown in live printing videos: Supplementary video 1 (Ref. 71) showing one pair of structures over a length of 500 μm in real time (15 fps) and supplementary video 2 (Ref. 71) showing repeated structures over a length of 2.5 cm captured at 60 fps over a period of 34 min, which is shown with 30× accelerated speed.
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