FDA penetrates the plasma membrane of cells due to its lipophilic nature. Once inside the cell, endogenous esterases cleave the diacetate groups, converting FDA into fluorescein, which is highly fluorescent and hydrophilic. Because dead cells lack functional esterases, they do not convert FDA into fluorescein, making this method a reliable indicator of cell viability. The fluorescein produced accumulates inside living cells and can be detected using a fluorescence microscope.
