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

ABSTRACT: 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

SUBMITTER: Chavez JC 

PROVIDER: S-EPMC6978660 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature