Micro-manipulation Methods Based On Cell Mechanical Properties

The micro-manipulation (MM) technique is one of vital approaches to operate cells. As the fast development of robotics and computer science, researches on automated and batch MM operations become the focus. However, as ignoring the mechanical harm to cells and the mechanical properties of cells, the presenting automated MM are only realized in some simple biological applications. In order to reduce the mechanical harm to cells and improve operational level, cell mechanical properties are introduced into the MM operations and the MM methods based on cell mechanical properties are studied using the traditional MM systems in this dissertation.This dissertation focuses on the online calibration methods of cell mechanical properties and the MM methods based on cell mechanical properties. In this dissertation, two vital cell mechanical properties, the mass and elastic modulus, are calibrated on line and used to achieve the quantitative control of the rotation angle (RA) in cell rotation manipulation. Base on that, a robotic oocyte enucleation process is achieved and then applied in domestic nuclear transplantation. Robotic production of the cloned embryos is achived. The main works of this dissertation are orgnized as follows:At present, cell weighing is usually achieved with specific devices and expensive instruments, which is not appropriate to be applied in MM. In this dissertation, a robotic cell weighing method based on the falling speed of the cell is achieved on the traditional MM system. In this method, the cell density is measured through the constant falling speed during falling process. Subsequently, the mass is calculated through the cell density. Experimental results demonstrate that the derived cell density is in accordance with the reported results in biological areas and our method is able to detect the1%variations of cell mass, which is about2.83×10-11kg.At present, the output pressure of the commercial pnuematic injector, which is usually used in MM, is influenced greatly by capillary effect and not accurate enough to be used to measure cell elasticity through the micropipette aspiration method. In this dissertation, a balance pressure model is presented and the influence of the capillary effect on aspiration pressure of the pneumatic injector is quantified for the first time. Subsequently, the aspiration pressureof the pneumatic injector is quantified using this model. Based on above technologies, a robotic elastic modulus calibration method is achieved on the traditional MM system and used to measure elastic modulus of the porcine oocytes/ferfilized eggs (about100μm) and mouse neusro spheres(about10μm).Based on above works, the derived cell mechanical properties are used to achieve quantitative control of the rotation angle (RA) in cell rotation. In this method, a minimum rotation force is derived through a force analysis on cells. Using the derived cell mechanical properties, the moving trajectories of the injection micropipette (IM) are derived and the quantitative control of the rotation angle is achieved using IM motion controllers. Experimental results demonstrate that our method can effectively improve the control accuracy of the RA and reduce the mechanical damages to cell.Further, combining the previous studies and the work in this dissertation, a robotic enucleation process for batch oocytes is achieved. Experimental results demonstrate that this enucleation process is able to operate oocytes with an average speed which is comparable to manual results with high proffessional skills. The success rate and the removing mass of the cytoplasm have advantages to manual method. Using this enucleation process in the sheep nuclear transplantation operation, the culture rate is comparable to that of manual method. Most importantly, several blastocysts are derived, which proves the feasility of the proposed method.