Three-dimensional motion measurements of free-swimming microorganisms using digital holographic microscopy

Abstract

A digital holographic microscopy was employed to measure the 3D motion of free-swimming microorganisms. A focus function was employed to quantify image sharpness for providing better depth-directional accuracy with a smaller depth-of-focus compared with employing the intensity method in determining the depth-directional position of spherical particles of various diameters. The focus function was 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 diameter-to-length ratios. The digital holographic microscopy technique developed in this study was found to be useful in measuring 3D swimming trajectories, velocities, and attitudes of hundreds of microorganisms simultaneously. It also exhibits exceptional depth-directional accuracy. The swimming characteristics of the solitary and chain-forming cells of marine dinoflagellate C. polykrikoides and P. minimum were measured using a digital holographic particle tracking velocimetry (PTV) technique and their dynamic behaviors were compared.