Precise 3D Tracking of Highly Non-Planar Eukaryotic Flagellar Beating Patterns Using Digital Holographic Microscopy.

Precise tracking of the rapid and complex 3D movement of eukaryotic flagella is important for understanding their roles in cellular motility, sensory functions, and resource acquisition. Yet, achieving accurate 3D kinematic reconstruction of flagellar beating patterns, particularly highly non-planar ones, remains challenging. Here we present holoV3C, a method based on Digital Holographic Microscopy (DHM) that allows precise, label-free 3D tracking of highly non-planar eukaryotic flagella with high temporal resolution. This algorithm leverages phase anomaly detection to provide a combination of high temporal and axial resolution, with 0.25 µm for beating mouse sperm flagella and down to 53 nm for polystyrene particles, across large sampling volumes in a computationally efficient manner. Algorithmic validation is performed by tracking mouse sperm flagella over time, capturing approximately 600 points along a single flagellum to achieve high axial resolution. Furthermore, we apply holoV3C to reconstruct the highly non-planar beating dynamics of the 200-nm-diameter flagellum of the protist Reclinomonas americana with a temporal resolution of 200 frames per second. By enabling 3D tracking of non-planar eukaryotic flagella, holoV3C can yield important insights to advance our understanding of flagellar dynamics, opening new avenues in the study of microorganism motility and its ecological roles.