Morphology and motility are essential criteria for assessing sperm viability. However, the human sperm head is small (∼3–4 μm) and requires a relatively high-magnification microscope objective, while the sperm flagella (∼45 μm) are poorly visible with complex 3D properties. Microscopic dynamic observation of intact sperm in 3D is challenging. Conventional inspection methods with a limited depth of field are inadequate for this issue. To provide a solution to this critical need, we develop pixelated polarization-based parallel phase-shifting digital holographic microscopy for the 3D dynamic observation of human sperm. Compared to conventional holographic imaging, this approach can effectively separate the object wavefront and avoid image quality degradation while fully exploiting the spatial bandwidth of the camera. We propose the use of the Stokes parameter reconstruction method to reconstruct the object wavefront and investigate the effect of the sampling interval on the system resolution by spectral analysis. The methodology achieves the retrieval of the 3D trajectory and motion parameters of sperm and reconstructs the sperm head orientation and the thin, highly-dynamic flagellum. The system allows for more comprehensive information on sperm motility and morphology, which is significant for male reproductive research. It also has significant potential for 3D dynamic observation of micro-organisms.

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