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Intracellular pH (pH  i  ) plays a crucial role in mammalian sperm physiology. However, it is a challenging task to acquire quantitative single sperm pH  i   images due to their small size and beating flagella. In this study, we established a robust pH  i   imaging system using the dual-emission ratiometric pH indicator, SNARF-5F. Simultaneous good signal/noise ratio fluorescence signals were obtained exciting with a green high-power LED (532 nm) and acquiring with an EM-CCD camera through an image splitter with two band-pass filters (550–600 nm, channel 1; 630–650 nm, channel 2). After  in vivo  calibration, we established an imaging system that allows determination of absolute pH  i   values in spermatozoa, minimizing cell movement artifacts. Using this system, we determined that bicarbonate increases non-capacitated human pH  i   with slower kinetics than in mouse spermatozoa. This difference suggests that distinct ionic transporters might be involved in the bicarbonate influx into human and mouse spermatozoa. Alternatively, pH  i   regulation downstream bicarbonate influx into spermatozoa could be different between the two species.

Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F