Micromanipulation is of critical importance in biomedical applications. Particularly

the precise 3 D positioning of pipette tip in micron-scale is a major topic of micromanipulation. The traditional algorithms function well for the horizontally-placed pipette. However

when facing the pipette that are placed obliquely

the traditional algorithms usually show the disadvantages of low precision

poor universality and susceptibility to influence of light source conditions. In order to improve positioning precision of pipette tip

a combined algorithm including the point scanning algorithm and automatic region of interest（ ROI） algorithm is proposed. The plane positioning precision is improved by the dual-end scanning of the pipette tip. The positioning precision is improved based on the automatic ROI algorithm combined with the Laplace variance focusing evaluation function. The experimental results show that the 3 D positioning error of the proposed method can be less than 1 μm when applied to the obliquely-placed pipette. Moreover

the proposed method overcomes the problems of low precision

poor universality and susceptibility to influence of light source conditions of traditional algorithms

lay a good foundation for precise micromanipulation.