Micropipette-Image Calibration Method Based on Micromanipulation System for Somatic Cell Microinjection

Research Background

Microinjection is a technique that employs a fine micropipette to inject a precise amount of genetic material, drugs, or other exogenous substances directly into cells or tissues. This technology plays a pivotal role in biomedical research, particularly in fields such as transgenics, gene targeting, animal cloning, human infertility treatment, and nuclease-guided genetic engineering. With advancements in automation technology, traditional manual microinjection has gradually evolved into automated operation modes. However, achieving accurate mapping between the observed 2D coordinates of the micropipette tip in microscope vision and the 3D positional data obtained from an external controller remains a significant technical challenge. Existing calibration methods often rely on motorized objectives or complex microscopic vision systems, which are not always fast or practical in real-world applications.

To address this challenge, Fei Pan and his team proposed a practical micropipette-image calibration method aimed at establishing precise mapping between the 2D pixel coordinates of the micropipette tip in the microscope field and the 3D spatial position data obtained from the external controller of an xyz micromanipulator. This study not only provides a new calibration method for somatic cell microinjection but also offers technical insights for other micromanipulation applications, such as micro-assembly.

Research Source

This paper was collaboratively authored by Fei Pan, Shuxun Chen, Liushuai Zheng, Shaohua Zhi, Xi Chen, and Dong Sun. The authors are affiliated with institutions such as Lingnan University and City University of Hong Kong. The paper was published in the IEEE Transactions on Automation Science and Engineering journal in 2025.

Research Content and Process

1. Micropipette-Image Calibration Method

The core of the study lies in proposing a micropipette-image calibration method based on the contact between the micropipette tip and the bottom of the culture dish. The specific process is as follows:

1. Coordinate System Definition: The study defines multiple spatial coordinate systems, including the pixel coordinate system (op, u, v) and the world coordinate system (o, x, y, z). Through the rotation and tilting of coordinate systems, the positional relationship of the micropipette tip in different coordinate systems is established.
2. Calibration Process: By moving the micropipette tip to three non-collinear contact points on the bottom of the culture dish, the 2D pixel coordinates and the 3D position data from the external controller are recorded at each contact point. Through matrix calculations, the mapping relationship between the pixel coordinates and the controller readings is established.
3. Error Analysis: The feasibility and stability of the method are verified through the analysis of calibration data.

2. Micropipette Tip-Breaking and Cell Preprocessing Techniques

To further improve the success rate of microinjection, the study also proposed two auxiliary techniques:

1. Micropipette Tip Breaking Technique: A low-cost acrylic ring is used to achieve controlled breakage of the micropipette tip, ensuring that the tip has a sufficiently small opening for single-cell microinjection. This method overcomes the drawbacks of dust accumulation and operational complexity in traditional methods.
2. Cell Preprocessing Technique: Fully adherent somatic cells are transformed into a semi-adherent state, increasing cell thickness and making them easier to puncture. This treatment method significantly improves the success rate and cell survival rate of microinjection.

3. Experimental Validation

The research team validated the effectiveness of the method through over 900 microinjection experiments on human dermal fibroblast (HDF) cells. The experimental results showed a success rate of 53.3% and a cell survival rate of 95.8%.

Research Conclusion

The study presents a practical micropipette-image calibration method, particularly suitable for micromechanical systems equipped solely with motorized micromanipulators but without motorized objectives. By combining the micropipette tip-breaking technique and the cell preprocessing technique, the research team successfully achieved efficient microinjection operations. This method not only has application value in somatic cell microinjection but also provides new solutions for other micromanipulation tasks.

Research Highlights

1. Innovative Calibration Method: The study proposes a micropipette-image calibration method based on the contact between the micropipette tip and the bottom of the culture dish, offering new technical support for precise microinjection.
2. Efficient Auxiliary Techniques: The tip-breaking technique and cell preprocessing technique significantly improve the success rate and cell survival rate of microinjection.
3. Experimental Validation: A large number of experiments validate the effectiveness and practicality of the method, providing technical references for future automated microinjection systems.

Research Significance

This study not only addresses the challenge of micropipette tip position calibration in microinjection but also provides new insights for the development of automated micromanipulation systems. In the future, this method can be integrated with other technologies to further optimize the precision and efficiency of microinjection systems, advancing biomedical research and clinical applications.