Long-term segmentation-free assessment of head–flagellum movement and intracellular calcium in swimming human sperm

Abstract

Human spermatozoa are the archetype of long-time self-organizing transport in nature, and critical for reproductive success. They utilize the coordinated head and flagellar movements to swim long distances within the female reproductive tract in order to find and fertilize the egg. However, to this date, long-time analysis of the sperm head-flagellar movements, or indeed those of 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). The computer-assisted segmentation-free method evaluates for the first time characteristics of the head movement and flagellar beating for up to 9.2 min. We demonstrate its powerful use by showing how releasing Ca²⁺ from internal stores significantly alters long-term sperm behavior. The method allows for straightforward generalization to other bio-imaging applications, such as Trypanosoma and bull sperm, or indeed to other flagellated microorganisms, also appealing to communities other than those from sperm biology.