Label-free 3D computational imaging of spermatozoon locomotion, head spin and flagellum beating over a large volume

Abstract

We report a high-throughput and label-free computational imaging technique that simultaneously measures in three-dimensional (3D) space the locomotion and angular spin of the freely moving heads of microswimmers and the beating patterns of their flagella over a sample volume more than two orders-of-magnitude larger compared to existing optical modalities. Using this platform, we quantified the 3D locomotion of 2133 bovine sperms and determined the spin axis and the angular velocity of the sperm head, providing the perspective of an observer seated at the moving and spinning sperm head. In this constantly transforming perspective, flagellum-beating patterns are decoupled from both the 3D translation and spin of the head, which provides the opportunity to truly investigate the 3D spatio-temporal kinematics of the flagellum. In addition to providing unprecedented information on the 3D locomotion of microswimmers, this computational imaging technique could also be instrumental for micro-robotics and sensing research, enabling the high-throughput quantification of the impact of various stimuli and chemicals on the 3D swimming patterns of sperms, motile bacteria and other micro-organisms, generating new insights into taxis behaviors and the underlying biophysics.

High-resolution 4-D acquisition of freely swimming human sperm cells without staining

“…In the last couple of years, several studies aimed at tracking the location of the entire flagellum in 3-D space and time (13)(14)(15). Silva-Villalobos et al (13) used an oscillating objective mounted in a bright-field optical microscope, covering a 16 µm depth at a rate of 5000 images per second, where the best flagellum focused sub-regions were associated to their respective z positions.…”

Multifocal imaging for precise, label-free tracking of fast biological processes in 3D

“…We have demonstrated the applicability of our technique for a label-free 3D reconstruction of human sperm's flagellar beat with unprecedented tempo-spatial precision. Common methods to characterize 3D flagellar beating of mammalian sperm involved combinations of light microscopy with a piezo-driven objective or digital holographic microscopy (DHM), also known as holographic phase retrieval 30,[37][38][39][40][41] . The piezo-driven method allows to acquire at Additionally, acquisition of planes takes place at different time points, requiring high-speed high-sensitivity cameras recording 5,000 images s -1 , that are very expensive and thus, restrict the availability of this technique to only few labs 41 .…”

Computer-assisted beat-pattern analysis and the flagellar waveforms of bovine spermatozoa

“…Whilst also of pertinence to a broad biological community, we anticipate that the data generated in this study (available as described in the accompanying data access statement) will also be of intense interest to the biophysical modelling community. Datasets generated with the same level of flagellar detail and scale have, to the best of our knowledge, been previously unavailable, with more of the distal flagellar tip being captured here than has been realised on large scales prior to this study, with the notable exception of the recent work of Daloglu et al [16] that applied sophisticated holographic imaging to capture beats in three dimensions. Easily-obtainable waveform data with the level of fidelity presented in this study has the potential to further the vast research area of heterogeneous population modelling, allowing the emergence of the next generation of evidence-based biophysical modelling for cell populations, spermatozoan behaviours in complex microenvironments, and more generally active matter physics.…”

“…A recent tool has been developed for this purpose with phase contrast microscopy by Gallagher et al [14], though it is limited due to its current inability to capture the distal region of the flagellum, despite the reported importance of the distal flagellum in motility mechanics [15]. Other recent works have similarly developed techniques to capture the beating flagellum, including the distal region, most notably the three-dimensional capture of the flagellar waveform achieved by Daloglu et al [16] with holographic imaging. However, compared to phase contrast microscopy, this is a highly-specialist imaging modality requiring custom circuitry together with sophisticated hardware and processing, whilst the former is already ubiquitous in theriogenology and andrology facilities [6,9], and also in the study of a wealth of eukaryotic monoflagellates [17].…”