Browsing by Subject "Microinjection"

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    Improved Computer Vision Algorithms for High-Throughput Targeting of Single Cells in Intact Tissue for Automated Microinjections
    (2021-10) O'Brien, Jacob
    Microinjection is a technique for organism-level and cellular-level manipulation of biological systems. The precise nature of microinjection permits the ability to target single cells in intact tissue which has enabled the study of cell-type related phenomena in development and disease progression. We envisioned the use of single-cellular microinjection as a tool for tagging cells with unique oligonucleotide barcodes that can be used during post-injection transcriptomic analysis to relate the transcriptomic reads with originally injected cells. For this process to be viable, we needed a system that was capable of precisely identifying the locations of cells in 3D tissue, assessing their feasibility for injection, and conducting rapid and large-scale microinjection into the identified cells. In this thesis, we report the development of such system. Our automated system uses computer vision algorithms to identify the 3D position of epifluorescent cells in intact tissue slices and assign them a quality metric to prioritize injections. The system guides a robotic micromanipulator to these cells and attempts injections while another computer vision algorithm and Kalman filter are used to improve the robot’s positioning accuracy. Additionally, cell impalement and cell filling detection algorithms were developed to evaluate injection success. We discovered, through a microinjection parameter sweep, an optimum combination of parameters to enable successful microinjection into a variety of cell types and tissue types. We used the optimized parameters to demonstrate automated tagging of single cells with a fluorescently labeled antibody targeting the nuclear pore complex proteins as a precursor step to fluorescence-based nuclei sorting and later transcriptomic analysis.
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    Universal robot for automated microinjection with applications in transgenesis and cryopreservation
    (2023-01) Joshi, Amey
    Microinjection is the process of injecting a small amount of solution into biological organisms at a microscopic level using a glass micropipette. It is a widely utilized technique with a wide range of applications in both fundamental research and clinical settings. However, microinjection is an extremely laborious and manual procedure, which makes it a critical bottleneck in the field and thus ripe for automation. In this thesis, we introduce a simple computer vision-guided robot that uses off-the-shelf components to fully automate the microinjection procedure in different model organisms. The robot uses machine learning models that have been trained to detect individual embryos on agar plates and serially performs microinjection at a particular site in each detected embryo with no human interaction. We deployed three such robots operated by expert and novice users to perform automated microinjections in zebrafish (Danio rerio) and Drosophila melanogaster. We conducted survivability studies to better understand the impact of microinjection on zebrafish embryos and the fundamental mechanisms by which microinjection affects zebrafish embryos. We were able to use the robot to examine the speed of the micropipette, the volume of the microinjectant, the micropipette geometry, and the rate of the volume delivered. These results helped us in determining the optimum settings for automated microinjection into zebrafish embryos. We used transgenesis studies to compare microinjection efficiency to manual microinjection utilizing optimum settings for automated microinjection. Further, we demonstrated that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and post-thaw survivability of cryopreserved embryos compared to manual microinjection, opening the door to large-scale cryo-banking of various aquatic species on an industrial scale. We anticipate that this robotic microinjection can be readily adapted to other organisms and applications.