Computer-assisted segmentation-free analysis for long-term assessment of flagellar beating in free swimming human spermatozoa: the role of emptying calcium stores

Microorganisms perform complex tasks and achieve biological function over long periods of time. Human spermatozoa are the archetype of long-time self-organizing transport in nature, and critical for reproductive success. They utilize the coordinated beating of their flagellum to swim long distances within the female reproductive tract in order to find and fertilize the egg. However, to this date, the long-time coordination of the sperm flagellum beating, or indeed other flagellated microorganisms, remain elusive due to limitations in microscope imaging and flagellar tracking techniques. Here, we present a novel methodology based on local orientation and isotropy of bio-image to obtain long-term kinematic and physiological parameters of individual free-swimming spermatozoa without requiring image segmentation (thresholding). This segmentation-free method allows for immediate evaluation of frequency and amplitude of the flagellar beat and head kinematic parameters for a duration of 9.2 min (limited by the cameras internal memory), corresponding to hundreds of thousands of flagellar beat cycles. We demonstrate the powerful use of this technique by examining how the release of Ca2+ from internal stores alters flagellar beating in the long-term, as Ca2+ is an essential regulator of flagellar beating. We report that increasing intracellular Ca2+ affected head and flagellar beat frequencies and amplitudes nonlinearly over long periods of time, commensurate with physiological conditions. The simplicity and robustness of the method may offer an advantageous alternative to segmentation-based methods, and allows for straightforward generalization to other bio-imaging applications, such as chlamydomonas, trypanosomes, c. elegans, or indeed flagellated microorganisms and slender-body organisms. Thus, this method may appeal to distant communities away from sperm biology.