A Practical Micropipette-Image Calibration Method for Somatic Cell Microinjection

This paper proposes a practical micropipette-image 2-D pixel and 3-D spatial coordination) calibration method indispensable to somatic cell microinjection, leveraging advancements in modern motorized micromanipulators. The method determines the depth information of the micropipette in the microscope field by assessing the contact between the micropipette tip and the bottom of the culture dish. It uses recoverable deformation upon contact as a criterion for precise positioning, ensuring the tip is on the dish’s bottom surface and in the microscope’s focus plane. Additionally, the paper introduces an on-the-spot method for breaking a micropipette tip and a preprocessing technique for somatic cells. The proposed micropipette tip-breaking method, using a low-cost acrylic ring, overcomes previous drawbacks and proves quick and user-friendly. The preprocessing technique converts fully adherent somatic cells into semi-adherent cells, increasing cell thickness for easier puncturing. Combining these techniques, the study validated the approaches through over 900 injections on human dermal fibroblast (HDF) cells, achieving a success rate of 53.3% and a survival rate of 95.8%. Note to Practitioners–This study was initiated to address the challenge of establishing accurate mapping relationships between observed 2-D coordinates of a micropipette tip in microscope vision and 3-D positional data obtained from an external controller of an XYZ micromanipulator. However, its relevance extends beyond somatic cell microinjection to other micromanipulation applications like micro-assembly. Common calibration methods involve motorized objectives or stereo microscopic vision, whereas our paper proposes a practical approach utilizing recoverable deformation upon tip contact with the dish bottom for precise positioning. The paper mathematically characterizes relationships between the pixel coordinate system and spatial coordinate systems, demonstrating problem-solving by ensuring the tip aligns with the dish’s bottom surface and the microscope’s focus plane. Although experiments affirm feasibility, refinement and testing in other micromanipulation applications are pending. Future research aims to enhance system modeling and calibration accuracy. © 2025 IEEE.