Automation of single biological cell surgery requires the location of organelles and cell structures to be determined to permit automated processing carried out in the cell surgery process. In this work, z-stack images of mouse embryos are used as model to develop image processing algorithms, to determine the centroid position (x,y,z) coordinates of embryo blastomeres. Transparency of embryos allow for a series of images along the vertical cell axis (z) to be obtained. Individual z-stack images are processed using 2D image processing steps to first segment, then estimate the centroid (x,y) coordinates of the blastomeres in the 2D image. Successive processing of all z-stack images then permits the centroid of the blastomeres to be determined in (x,y,z) coordinates. Image processing-based calibration allows PZD micropipette to move to the computed centroid position with PBVS control. These algorithms are experimentally verified with mouse embryos at the 2 blastomere stage of development.