A prominent technical barrier when imaging swimming sperm is capturing a singular sperm cell's head and tail position simultaneously at a high resolution to understand their relationship in different stages of the sperm tail beating cycle. This is due to the sperm's high beating frequency, rotational movement, and the large difference in diameter between the head and tail. These intricacies increase the complexity of determining the position of a dynamic subcellular structure in the sperm neck, such as the centriole. We have developed a way to obtain this information by snap freezing mobile sperm at different stages of the sperm tail beating cycle and then analyzing them with super-resolution microscopy. This method captures the position of both the sperm head and tail at the microscale and centriolar substructure details at the nanoscale. This chapter describes the detailed procedures for the selection, preparation, antibody staining, 3D N-STORM imaging, and image quantification of bovine spermatozoa.
Keywords: Centriole; Density gradient technique; Immunofluorescence staining; STORM; Spermatozoa; Swim-up technique.
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