PROJECT SUMMARY Biomedical applications in both research and clinical settings rely on microinjection protocols that require using a hollow microcapillary needle to deliver foreign substances (e.g., nucleic acids, proteins, viruses, cells, and nanoparticles) directly into biological targets (e.g., cells, embryos, and tissues). This transgenic engineering technique has driven advances in fundamental research in areas including cell, systems, and developmental biology, stem cell gene manipulation, and human disease prevention modeling, as well as for medical applications including in vitro fertilization (IVF) cycle therapy, pre-implantation genetic diagnosis, and intraocular injection. However, the industry standard microneedles (ISNs) used for microinjection in research settings are typically produced lab-by-lab by physically “pulling” apart transparent glass capillary tubes, which results in needle variability. The inconsistencies can negatively affect experimental rigor, reproducibility, productivity, and microinjection efficacy. Thus, the ability to enhance and expand the capabilities of the microneedles that enable microinjection protocols could significantly impact diverse biomedical fields and applications. We hypothesize that by leveraging well-established “pyrolysis” post-processing strategies for enhancing the mechanical properties of DLW-printed 3D microstructures, we can achieve mechanically robust microinjection needles—based on our pending-patented MSP design—that simultaneously address all the pain points of ISNs. This proposal will systematically evaluate the utility of the mechanically robust 3D-printed microcapillary needles versus ISNs with regard to key quantitative metrics of performance underlying material delivery into zebrafish embryos by: 1) establishing and characterizing pyrolysis protocols for DLW-printed 3D microneedles, 2) Interrogating the mechanical properties of pyrolyzed 3D microneedle prototypes in vitro, and 3) evaluating microinjection efficacy of pyrolyzed DLW-printed 3D microneedle prototypes versus ISNs using live zebrafish embryos in vivo. These innovative, geometrically sophisticated, functionally advantageous microneedle with robust mechanically properties hold unique promise to allow for fast, accurate, and consistent microinjections with minimal damage to injection targets (e.g., embryos).