Miano, F. | Search bioRxiv

bioRxiv2025

Precise 3D Tracking of Highly Non-planar Eukaryotic Flagellar Beating Patterns using Digital Holographic Microscopy

Precise tracking of the rapid and complex three-dimensional 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 m 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.

biophysics↗

bioRxiv2023

FORAGING MECHANISMS IN EXCAVATE FLAGELLATES SHED NEW LIGHT ON THE FUNCTIONAL ECOLOGY OF EARLY EUKARYOTES

The phagotrophic flagellates described as typical excavates have been hypothesized to be morphologically similar to the Last Eukaryotic Common Ancestor and understanding the functional ecology of excavates may therefore help shed light on the ecology of these early eukaryotes. Typical excavates are characterized by a posterior flagellum equipped with a vane that beats in a ventral groove. Here, we combined flow visualization and observations of prey capture in representatives of the three clades of excavates with computational fluid dynamic modelling, to understand the functional significance of this cell architecture. We record substantial differences amongst species in the orientation of the vane and the beat plane of the posterior flagellum. Clearance rate magnitudes estimated from flow visualization and modelling are like that of other similarly sized phagotrophic flagellates. The interaction between a vaned flagellum beating in a confinement is modelled to produce a very efficient feeding current at low energy costs, irrespective of the beat plane and vane orientation and of all other morphological variations. Given this predicted uniformity of function, we suggest that the foraging systems of typical excavates studied here may be good proxies to understand those potentially used by our distant ancestors more than 1 billion years ago. SignificanceHuman sperm reminds us of our ancestry: flagellates, unicellular organisms equipped with a flagellum. The last common eukaryotic ancestor (LECA) was a flagellate. Phylogenetic analyses suggest that Excavates, an assemblage of flagellates, are the living organisms most similar to LECA. They have distinct characteristics in common: a ventral groove within which a vaned flagellum is beating. We show how the shared morphology and foraging behavior among 3 excavate clades is fluid dynamically efficient. A similar flagellar arrangement, potentially homologous to that found in the excavates, is found among flagellates from other deep branches of the eukaryotic tree, suggesting that the typical excavate foraging system studied here may have been used by our distant ancestors more than 1 billion years ago.

ecology↗