| Summary: | Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technology used in infertility treatment, where a single sperm is selected and immobilized using a glass injection pipette and is inserted directly into the cytoplasm of an oocyte under the microscope. Auto-alignment of the injection pipette is a prerequisite for any proposed automated oocyte injection procedure. In this paper, an auto-alignment procedure has been proposed. This technique requires the positioning of the injection and holding pipettes in the three orthogonal axes under microscopy which is complex. The existing proposed methods are system-specific and require appropriate algorithms. In this paper, 12 commonly used focusing algorithms were evaluated to verify the optimal one for the ICSI application. These algorithms were assessed by measuring focusing accuracy, range, the number of false maxima, and the width of the curve. The focus level for each pipette is calculated by the algorithm using focus measure functions (FMA). The Fibonacci search algorithm is employed for controlling the z-axis of the motorized stage to obtain the focal plane of the injection and holding pipette. The experimental results verified that the Brenner gradient has demonstrated the highest overall performance for injection pipette focalization, while the energy of gradient has presented a highest overall performance for holding pipette focalization.
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