**5. Summary and Perspectives**

The recent advances in molecular imaging techniques using cryo-EM, XFEL, and synchrotron facilities necessitates the precise and controlled delivery of mixed solutions. Microfluidic technology has shown promise in addressing the sample delivery needs for molecular imaging technology over recent decades. Here, we have reviewed the recent advances in the emerging field of integrated mix-and-jet microfluidic sample delivery devices.

We introduced the main parameters required for the design of these integrated devices. The nozzle component is mainly designed based on the GDVN principle and integrated into the microfluidic device to generate free-standing liquid jets. The primary dimensionless parameters to be considered for the nozzle design and characterisation of the jet are *We* and *Re*. Passive micromixers are commonly used to trigger biomolecular reactions, taking advantage of chaotic advection and rapid millisecond mixing. The main dimensionless parameters to be considered for the design of a passive mixing component are *Re* and *Pe*, whilst *ηmixing* can characterise the mixing in the mixer microchannel. Additionally, we critically reviewed the techniques used for the fabrication of the mix-and-inject devices. Conventional capillary-based methods for the fabrication of the sample delivery devices are laborious and irreproducible, providing only limited versatility to integrate complex passive micromixers. Numerous techniques for the fabrication of chip-based microfluidic mix-and-inject devices were reported to replace the previous capillary-based techniques. Most of the chip-based planar methods enable the fabrication of rigid and chemically inert devices whilst taking advantage of the design freedom, high resolution, and reproducibility. Recently, 3D printed mix-and-jet microfluidic devices have shown great promise for XFEL single-particle imaging and SFX studies. The new technology facilitates fast and low-cost fabrication of fully 3D mixer and nozzle components that outperform both capillary and on-chip sample delivery devices. Furthermore, we summarised the standard experimental

techniques used for the characterisation of both mixing and jetting. For these measurements, both high-speed optical imaging and fluorescent signal analysis were used.

Incorporating GDVN nozzles with microfluidics technology is still a new concept that will open up a host of new applications in many areas, especially in the biological and life sciences. Currently, most of the published references in this field are proof-of-concept of mix-and-inject experiments in which new device architectures and designs are often introduced. In the near future, we can expect to see more reports describing innovative designs and solutions to apply these devices to a range of different fields, including fundamental chemistry and physics, polymer fabrication, the study of the kinetics of nanoparticles, and biomolecular imaging.

**Author Contributions:** M.H. conceived of the presented idea, developed the structure of the paper, and wrote the manuscript. B.A. and E.B. supervised the work. All authors discussed the review paper and contributed to the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Australian Research Council Centre of Excellence in Advanced Molecular Imaging, grant number CE140100011.

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Acknowledgments:** This work was performed in part at the Melbourne Centre for Nanofabrication (MCN) in the Victorian Node of the Australian National Fabrication Facility (ANFF). The authors would like to acknowledge the support of the Australian Research Council (ARC) Centre of Excellence in Advanced Molecular Imaging.

**Conflicts of Interest:** The authors declare no conflict of interest.

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