Artifacts Generated by the 3D Rotation of a Freely-Swimming Human Sperm in the Measurement of Intracellular Ca2+

Intracellular calcium [Ca2+]i is key to many sperm functions including swimming, locating the egg, and fertilizing it. However, the 3D motion of the cell complicates measuring the [Ca2+]i of freely-swimming cells, mainly because a sperm rotates on its axis of movement as it swims (head and flagellum rolling), which may add spurious fluctuations (artifacts) to the fluorescence images. Here we implement an experimental device and software that for the first time allow quantifying the effect of the head spin on the [Ca2+]i signal. The device is a 3D + t multi-focal-plane optical microscope that permits simultaneous recording of bright-field and fluorescence images. The scripts we developed allow measurement of the head’s spin time series from the bright-field images and the [Ca2+]i signal from the fluorescence images. We found that the fluorescence signal of cells labeled with a [Ca2+]i-insensitive dye significantly resemble the rotation signal, while cells with a calcium-sensitive dye have a high-frequency harmonic related to the beat of the flagellum.