![]() The crystal is subsequently destroyed (after each measurement), necessitating continuous replenishment of sample into the X-ray beam. This has extended X-ray crystallography to time-resolved analysis of proteins using micrometer-sized crystals and smaller. The method greatly increases the exposure of a crystal beyond conventional limits, and without the need for cryogenic cooling, by using femtosecond-duration X-ray pulses that terminate before the onset of radiation damage 8. It has allowed the observation of radiation-sensitive structures at the atomic scale 5, 6, 7. Serial femtosecond crystallography (SFX) at X-ray free-electron lasers seeks to extend the sensitivity and applicability of X-ray diffraction to structural biology. The ability to create free-form features with submicron accuracy promise performance improvements for microfluidic engineering. Originally pioneered for micro/nano-optics applications 2, the utility of 2pp for microfluidic engineering remained limited as devices required several hours of print time per chip 3, 4. In particular, two-photon stereolithography (2pp) is one of the few 3D printing methods that achieve free-form geometries with submicron precision. While numerous 3D printing techniques have been explored for constructing 3D microfluidics, a trade-off between resolution and throughput imposes a practical resolution limit for such 3D microsystems 1. Microfluidic chips are fabricated through two-dimensional (2D) lithography and increasingly by additive techniques, commonly referred to as three-dimensional (3D) printing 1. Microfluidic precision and miniaturization revolutionized the microscale manipulation of reagents and cells that nowadays have numerous applications in the natural sciences, engineering, and industrial applications. This technology has the potential to permit ultracompact devices and performance improvements through 3D flow optimization in all fields of microfluidic engineering. Such devices can be printed in minutes by locally adjusting print resolution during fabrication. Also, aberration-free in operando X-ray microtomography is introduced to study efficient equivolumetric millisecond mixing in channels with 3D features integrated into the nozzle. By integrating an additional orifice, we implement a low consumption flow-focusing nozzle, which is validated by solving a hemoglobin structure. We achieve submicron jets with speeds exceeding 160 m s −1, which allows for the use of megahertz XFEL repetition rates. We demonstrate this technique by tailoring microfluidic nozzles and mixers for time-resolved structural biology at X-ray free-electron lasers (XFELs). To advance microfluidic integration, we present the use of two-photon additive manufacturing to fold 2D channel layouts into compact free-form 3D fluidic circuits with nanometer precision.
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