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

Human spermatozoa are the archetype of long-term self-organizing transport in nature and are critical for reproductive success. They utilize coordinated head and flagellar movements to swim long distances within the female reproductive tract in order to find and fertilize the egg. However, to date, long-term analysis of the sperm head–flagellar movements, or indeed those of other flagellated microorganisms, remains elusive due to limitations in microscopy and flagellar-tracking techniques. Here, we present a novel methodology based on local orientation and isotropy of bio-images to obtain long-term kinematic and physiological parameters of individual free-swimming spermatozoa without requiring image segmentation (thresholding). This 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 studies of bull sperm and Trypanosoma, or indeed of other flagellated microorganisms – appealing to communities other than those investigating sperm biology.