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Monitoring labile Zn2+ homeostasis is of great importance for the study of physiological functions of Zn2+ in biological systems. Here we report a novel ratiometric fluorescent Zn2+ sensor, CPBT, which was constructed based on chelation-induced alteration of FRET efficiency. CPBT was readily cell membrane permeable and showed a slight preferential localization in the endoplasmic reticulum. With this sensor, 3D ratiometric Zn2+ imaging was first realized in the head of zebra fish larvae via Z-stack mode. CPBT could track labile Zn2+ in a