Real-Time Phosphate Sensing in Living Cells using Fluorescence Lifetime Imaging Microscopy (FLIM)

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.