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A digital holographic microscope is employed to measure the 3D motion of free-swimming microorganisms. The focus function used to quantify image sharpness provides a better depth-directional accuracy with a smaller depth-of-focus compared with the intensity method in determining the depth-directional position of spherical particles of various diameters. The focus function is then applied to measure the 3D positions of free-swimming microorganisms, namely dinoflagellates C. polykrikoides and P. minimum. Both automatic segmentation and proper selection of a focus function for a selected segment are important processes in measuring the positional information of two free-swimming microorganisms of different shapes with various width-to-length ratios. The digital holographic microscopy technique improved in this work is useful for measuring 3D swimming trajectories, velocities and attitudes of hundreds of microorganisms simultaneously. It also exhibits exceptional depth-directional accuracy.

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