Phosphate ions play important roles in signal transduction and energy storage in biological systems, However, robust chemical sensors capable of real-time quantification of phasphate anions in live cells have not been developed. The fluorescein derivative dye 9- [1-(2-methyl-4-medioxyphenyl)]-6-hydrox-3H-xanthen-3-one (2-Me-4-OMe TG) exhibits the characteristic excited-state proton-transfer (ESPT) reaction of xanthenic derivatives at approximately physiological pH resulting in the dependence of the dyes, nanosecond fluorescence: decay time on the phosphate buffer concentration. This allows the 2-Me-4-OMe, TG dye to. be used with fluorescence lifetime. imaging; microscopy (FLIM) as a real-time phosphate intracellular intracellular sensor cultured cells. This methodology as allowed the time course Of cellular differentiation of MC3T3-E1 murine preosteoblast cells to be measured on the basis of the decrease in the decay time of 2-Me-4-OMe These Changes were phosphatase activity in the extracellular medium as a marker of the differentiation process.